ABSTRACT Advances in characterization of the class I antigen presentation pathway over the last decade have taught us that antigen processing, peptide transport, peptide trimming, peptide selection, and peptide loading are critical events for the development of optimal peptide repertoires presented by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). These events influence the tight binding of peptides needed to generate conformationally stable MHC I/peptide complexes. Evidence has been provided that changes in the overall quality of the MCH I peptide repertoire can have profound effects on CTL responses. Therefore, studies that characterize basic biological events in MHC I maturation are essential not only for advances in fundamental principles of antigen processing and presentation, but also to better comprehend how these maturation events modulates immune responses. We have studied the molecular and structural basis of mechanisms of peptide loading and selection by MHC I for more than 20 years. Recently, the endoplasmic reticulum-aminopeptidases associated with antigen processing (ERAP1, ERAP2, and ERAP1,2 heterodimer; referred to as ERAP) have emerged as key proteins for influencing formation of the MHC I peptide repertoire. To date, our understanding of how ERAP functions as a peptide editor is however obscure. Similarly, the question of how ERAP1 and ERAP2 polymorphisms play a role in chronic autoimmune diseases such as HLA-B*27-associated ankylosing spondylitis (AS), and how ERAP1 and ERAP2 interact with disease-associated MHC I molecules, still remain unclear. In this application, we will use a comprehensive approach to shed some light on the function of ERAPs and on the molecular crosstalk between ERAPs and MHC I molecules. Using a cell-free system composed of ERAPs, MHC class I molecules, and synthetic and natural N-terminally extended peptides, and in combination with biochemical, molecular, and crystallographic techniques, we will characterize the function of ERAPs towards free and MHC I-bound peptides (Aim #1); elucidate the functional links between ERAP1, HLA-B*27, and AS (Aim #2); and determine the x-ray crystal structure of an HLA-B*0801/precursor complex, with and without ERAP1 (Aim #3). Overall, our study is fundamentally significant because it will provide new knowledge to better understand how ERAPs function and influence formation of the MHC I peptide repertoire, novel insights into molecular events underlying the pathogenesis of AS, and will reveal the molecular interaction between ERAP1 and MHC I molecules. The medical relevance of our studies is immense.